Novel Dried Blood Spot Mass Spectrometry Functional Assays for Lysosomal Acid Lipase and N-acetylgalactosamine-sulfate Sulfatases for Use in Newborn Screening

 描述(由申请人提供)：人类胆固醇运输途径的缺陷可能导致两种最终致命的隐性疾病(沃尔曼氏病和胆固醇酯储存病，或CESD)。负责的酶，溶酶体酸性脂肪酶，或LAL，不能执行其胆固醇酯水解功能，导致Wolman病患者的肝功能严重下降，血清中胆固醇和甘油三酯水平迅速上升，CESD患者血管中动脉粥样硬化性疾病的发病率在成年早期增加。然而，对于这两种疾病中的任何一种，都没有推荐的或通用的诊断方法，导致许多漏诊病例。通过使用特定的LAL底物或现有的LAL不可逆抑制剂，我们将能够特异性地检测干血斑中的LAL酶活性。同样，开发一种更灵敏的DBS方法来检测更为罕见的粘多糖病(MPS)IVA和VI型，将极大地有助于筛查和诊断由于这些酶的基线活性较低而导致的缺陷。使用干血斑(DBS)是白细胞中发现的酶的一种低成本、相对长寿的来源。质谱仪将用于量化子离子碎裂(或MS/MS扫描)产生的裂解底物的数量，为酶功能的微小变化提供高灵敏度，这对于最大限度地减少临床环境中的假阳性至关重要。的发展。 MS/MS干血斑点检测LAL缺乏症以及改进的MPS IVA和VI MS/MS DBS检测将为新生儿筛查中心提供一种有效识别潜在病例的手段，这些病例需要立即干预和可能的终身治疗，以保护这些受影响儿童的生命。

 DESCRIPTION (provided by applicant): Defects in the human cholesterol transportation pathway can create two ultimately fatal recessive diseases (Wolman's disease and Cholesterol Ester Storage disease or CESD). The responsible enzyme, lysosomal acid lipase or LAL, is unable carry out its cholesterol ester hydrolysis function, resulting in a severe decline in liver function for Wolman's disease patients and a rapid increase in both cholesterol and triglyceride levels in blood serum with increased incidence of atherosclerotic disease in CESD patients' vessels by early adulthood. Yet, no recommended or universal diagnostic assay exists for either of these two diseases, resulting in many missed cases. By using a specific LAL substrate or an existing irreversible inhibitor of LAL, we will be able to specifically assay LAL enzymatic activit in dried blood spots. Likewise, the development of a more sensitive DBS assay for the more rare mucopolysaccharidoses (MPS) types IVA and VI will greatly benefit screening and diagnosing deficiencies due to the low baseline activity of these enzymes. The use of dried blood spots (DBS) is a low cost, relatively long-lived source of the enzymes found in leukocytes. Mass spectrometry will serve to quantify the amount of cleaved substrate from daughter ion fragmentation (or MS/MS scans), providing a high sensitivity for even small changes in enzyme function, which is critical for minimizing false positives in clinical settings. The development of an MS/MS dried blood spot assay for LAL deficiencies as well as improved MPS IVA and VI MS/MS DBS assays will provide newborn screening centers a means to efficiently identify potential cases that require immediate intervention and potentially life-long therapy that will protect the lives of these affected children.